APPLICATION - Hitachi Centrifuges

No.103
APPLICATION
October 2000
Separation of lipoprotein from serum with the new micro ultracentrifuge at million x g
CS150GXL/CS150GX micro ultracentrifuges and S140AT fixed angle rotor
Example:
Separation of lipoprotein (VLDL, LDL and HDL) from human serum in only 270 minutes
Lipoproteins that are included in serum have a close relation to metabolism of lipid, and they are noticed
in the research field on hyperlipemia. This time, lipoproteins VLDL, LDL and HDL were separated from
normal human serum with the newly developed fixed angle rotor that has the world’s highest RCF of
1,050,000 x g.
1. Equipment used
Centrifuge: Hitachi CS150GXL preparative micro ultracentrifuge
Rotor: S140AT fixed angle rotor
(Max. speed: 140,000 rpm, max. RCF: 1,050,000 x g, max. capacity: 2 ml x 10 tubes)
Tube: 1PC tubes
2. Procedures and results
FAT RED 7B was added to the sample in each right tube below for easier visual checking.
(1) Separation of VLDL (Density<1.006 g/ml)
Centrifugation
Serum
600
Add 300 micro
litters
of
specific gravity
liquid A on the
layer.
140,000 rpm
50 min., 16
degrees
centigrade
ACCEL:5
DECEL:7
micro litters
Note that the above VLDL fraction includes chylomicron.
VLDL
LDL, HDL,
albumin,
etc.
(2) Separation of LDL (1.006 g/ml<Density<1.063 g/ml)
Centrifugation
Remove
the
300
micro litters top layer
of (1) (VLDL fraction).
Add 300 micro
litters
of
specific
gravity liquid B
and mix them
gently
140,000 rpm
80 min., 16
degrees
centigrade
ACCEL:9
DECEL:7
LDL
HDL,
albumin,
etc.
Note that the above LDL fraction includes IDL.
(3) Separation of HDL (1.063 g/ml<Density<1.21 g/ml)
HDL
Centrifugation
Remove the 300 micro
litters top layer of (2)
(LDL fraction).
140,000 rpm
140 min., 16
degrees
centigrade
ACCEL:9
DECEL:7
Add 300 micro
litters
of
specific
gravity liquid C
and mix them
gently
Albumin,
etc.
Specific gravity liquid A (Density:1.006 g/ml):
Put 11.40 g of NaCl and 0.1g of EDTA-2Na in a 1,000 ml measuring flask. Add 500 ml of
distilled water and 1 ml of 1N NaOH, and mix them well until they are dissolved. Add distilled
water up to 1,000 ml and then add additional 3 ml of distilled water. (NaCl: 0.195 mol)
Specific gravity liquid B (Density:1.182g/ml):
Add 24.98 g of NaBr to 100 ml of the specific gravity liquid A. (NaCl: 0.195 mol, NaBr: 2.44 mol)
Specific gravity liquid C (Density:1.478g/ml):
Add 78.32 g of NaBr to 100 ml of the specific gravity liquid A. (NaCl: 0.195 mol, NaBr: 7.65 mol)
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